Targeting TBL1X induces DNA damage in MCL cells. (A) Immunoblot analyses of MCL cell lines (JeKo-1, SP-53, and Granta-519) treated with tegavivint (IC50, 12-18 hours) compared with dimethyl sulfoxide (DMSO) control for quantification of γH2AX formation (Ser-139 phosphorylation). Immunoblot analyses for γH2AX formation in (B) tegavivint-treated (18-hour, 0-100 nM) primary MCL patient samples (n = 4) and (C) MCL cell lines (n = 2) subjected to TBL1X shRNA KD with specific constructs (n = 2; sh425 and sh525) or EV control. (D) Immunofluorescence for γH2AX (red) foci with quantification (number of γH2AX per cell) in tegavivint-treated (IC50, 12 hour) MCL cell lines (n = 2) compared with DMSO control; nuclei are stained with DAPI (blue). Photomicrographs were taken at original magnification ×200 or ×600 (inset). (E) γH2AX immunohistochemistry with quantification (digital analysis for positive pixels) in the tumors of a subset of mice from the FC-muMCL1 study (Figure 2D) euthanized at a predetermined time point (day 14 after engraftment, after n = 4 tegavivint treatments). Brown chromogen (3,3'-diaminobenzidine or DAB) indicates positive γH2AX immunostaining; nuclei were counterstained blue-purple with Harris hematoxylin. Photomicrographs were taken at original magnification ×600; black scale bars, 20 μm. (F) Immunoblot analyses of MCL cell lines (JeKo-1, SP-53, and Granta-519) treated with tegavivint (IC50, 12-18 hours) compared with DMSO control for quantification of DDR Chk1 and Chk2 kinase activation (phosphorylation) and CDT1 levels. Tegavivint IC50 concentrations = 95 nM for JeKo-1, 65 nM for SP-53, and 200 nM for Granta-519. DAPI, 4′,6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; hr, hour; NT, no treatment; NQ, not quantifiable. ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001.
